DESCRIPTION: Genetic regulation is essential to survival in all living organisms. Protein binding to specific target DNA sequences is a primary mechanism for control of transcription initiation in both prokaryotic and eukaryotic organisms. Protein-DNA recognition in turn is modulated by binding partnerships with other molecules, including both small metabolites and homo- and heteromeric protein interactions. The prototypic prokaryotic negative transcriptional regulator is lac repressor, a homotetrameric protein that prevents initiation of mRNA encoding the lactose metabolic enzymes unless substrate is available in the environment. This protein is a member of a larger family of proteins that participate in transcriptional regulation in Escherichia coli. The Ultrabithorax (UBX) protein is a member of the homeotic gene family, conserved among a broad range of species, in which each member contains a DNA recognition motif known as the homeodomain. UBX protein is involved in specifying segmental identity during development in Drosophila by altering transcription of specific genes. Differences in regulation by UBX protein (activation vs. repression) may be derived at least in part from homotropic and heterotropic associations with other proteins. The goal of this project period is to decipher structural contributions to allostery, stability, activity modulation, and assembly (both homo- and heteromeric) in these two regulatory proteins. The mechanisms of allostery and stability in the lac repressor will be examined by site-specific mutagenesis with residues selected for substitution based on the recently solved crystallographic structures of the core domain of this protein and the purine holorepressor complex. Binding properties of the N-terminal helix-turn-helix domain of lac repressor and homeodomain of UBX protein are modulated by association with the remainder of the protein structure. The properties of covalently linked helix-turn-helix domains alone and complexed with DNA will be examined. In addition, the regions of the UBX protein that diminish homeodomain affinity for target DNA sites will be identified by biochemical and genetic methods. Regions and specific residues required for homologous protein-protein assembly will be examined in both lac repressor and UBX protein. The set of residues necessary to generate a subunit interface will be explored by assembly of a hybrid protein consisting of the N-terminal helix-turn-helix DNA binding domain of lac repressor, periplasmic sugar binding protein (normally monomeric), and the C-terminal domain of repressor required for dimer-dimer association. Structural determinants for heterologous protein-protein association will be explored in these regulatory proteins, and partners for UBX protein will be identified. The information generated by these experiments will expand significantly our understanding of the structure and function of these essential genetic regulatory proteins. Because of structural homology with other members of their respective protein families, the results on lac repressor and UBX proteins will provide information for designing regulatory proteins and will yield general insights into principles of protein structure and function.